Two apparent human endothelial cell growth factors from human hepatoma cells are tumor-associated proteinase inhibitors

Two polypeptides from secretory products of human hepatoma cells were isolated and characterized on the basis of their stimulation of maintenance and growth of human endothelial cells in serum-free cell culture. Both factors were purified to homogeneity by a combination of reverse-phase, ion exchange, and molecular filtration high performance liquid chromatography. One factor (endothelial cell growth factor (ECGF-2a) had Mr approximately 6,500 and pI near 6. The second (ECGF-2b) had Mr = 27,000 and a pI below 4.0. Both ECGF-2a and ECGF-2b exhibited single NH2-terminal sequences. The first 25 NH2-terminal residues of ECGF-2a and the first 49 residues of ECGF-2b were determined by gas-phase microsequencing. All clearly determined residues of ECGF-2a were identical with human pancreatic secretory trypsin inhibitor. All assignable residues of ECGF-2b were identical with urinary glycoprotein proteinase inhibitor (HI-30/EDC1). Both proteins are absent or at low levels in normal plasma and urine, but appear during acute inflammatory disease and cancer. Amino acid composition of ECGF-2a and ECGF-2b was also similar to human pancreatic secretory inhibitor and HI-30/EDC1, respectively. Both ECGF-2a and ECGF-2b inhibited bovine pancreatic trypsin (2 micrograms/ml) by 50% at 750 ng/ml. ECGF-2a and ECGF-2b stimulated endothelial cell number at a half-maximal dose of 50 ng/ml (8 nM) and 80 to 130 ng/ml (5 to 9 nM) protein, respectively. When assayed under identical conditions, no effect of either factor on human smooth muscle cells, human hepatoma cells, or human, rat, and mouse fibroblasts could be detected.     

Separation of plasmid-mediated extended spectrum β-lactamases by fast protein liquid chromatography (FPLC system)

We have devised a reliable procedure for the separation of three beta-lactamases of isoelectric focusing points (pI), 5.4, 6.5, and 7.9 by Fast Protein Liquid Chromatography (FPLC System). All of these enzymes were transferable and originated from a ceftazidime and cefotaxime resistant Klebsiella pneumoniae isolated in Bombay, India. The complete separation of the enzymes, achievable by this method, allowed each of the different individual beta-lactamases to be characterized biochemically. This analysis revealed that the enzymes of pI 6.5 and pI 7.9 hydrolysed ceftazidime and cefotaxime, and were responsible for the resistance of K. pneumoniae, and its Escherichia coli J53-2 transconjugant to third generation cephalosporins. The enzyme of pI 5.4 was the TEM-1 beta-lactamase. The beta-lactamase of pI 7.9 appears quite different from any previously reported third generation cephalosporin hydrolysing beta-lactamase, and consequently given the preliminary designation DJP-1. This is also the first example of extended spectrum hydrolysing beta-lactamases found in Asia.     

A synthetic presequence reversibly inhibits protein import into yeast mitochondria

We show that a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence blocked import of pre-subunit IV into yeast mitochondria. The 22-residue peptide pL4-(1-22) did not alter the electrical potential across the mitochondrial inner membrane (the delta psi). Inhibition of import was reversible and could be overcome by the addition of increased amounts of precursor. Two other peptides, pL4-(1-16) and pL4-(1-23), which correspond to, respectively, the N-terminal 16 and 23 residues of the same presequence, also blocked import of pre-subunit IV. However, pL4-(1-16) was a much weaker inhibitor of import, while the inhibitory effect of pL4-(1-23) was due to its ability to completely collapse the delta psi. pL4-(1-22) seems to be a general inhibitor of mitochondrial import, in that it also blocked uptake of several other proteins. These included the precursors of the yeast proteins cytochrome c oxidase subunit Va, the F1-ATPase beta subunit, mitochondrial malate dehydrogenase, and the ATP/ADP carrier. In addition, uptake of two non-yeast precursor proteins (human ornithine transcarbamylase and a cytochrome oxidase subunit IV-dihydrofolate reductase fusion), was also blocked by the peptide. Subsequent studies revealed that pL4-(1-22) did not block the initial recognition or binding of proteins to mitochondria. Rather, our results suggest that the peptide acts at a subsequent translocation step which is common to the import pathways of many different precursor proteins.     

Electrotransfer of basic proteins from nondenaturing polyacrylamide acid gels to nitrocellulose: detection of enzymatic and inhibitory activities and retention of protein antigenicity.

We have developed a method for electrotransfer of strongly basic proteins (lysozyme, pI 11; mucus proteinase inhibitor, pI greater than 10; bovine pancreas trypsin inhibitor; pI 10.5; human leukocyte elastase, pI greater than 9) from nondenaturing acid gels (pH 4.5) to nitrocellulose sheets. Buffers were those used in a discontinuous system for transfer from sodium dodecyl sulfate (SDS)-containing polyacrylamide gels with one modification in the cathode buffer which contained 0.1% SDS. This method was compared to electrotransfer performed in 0.7% acetic acid. The basic proteins studied, which were positively charged in the gel, formed with SDS negative complexes which migrated toward the anode and were efficiently transferred to the nitrocellulose. Moreover, their biological properties were preserved: inhibitory activity, enzyme activity, and antigenicity. This method is advantageous because it is simple, is sensitive, and can be applied to various biological fluids to detect inhibitors, enzymes, and other proteins which have a basic character, after electrophoretic separation under their native forms.     

A 75-kDa Na+,K+-ATPase competitive inhibitor protein isolated from rat brain cytosol binds to a site different from the ouabain-binding site.

A Na+,K+-ATPase inhibitor protein has been purified to homogeneity from rat brain cytosol by ammonium sulphate precipitation, DEAE anion-exchange chromatography and hydroxyapatite adsorption column chromatography. The purified protein migrates as a single polypeptide band of 75 kDa on 7.5% SDS/PAGE. Amino acid composition data shows the presence of a high number of acidic amino acids in the molecule in relation to the pI value of 4.6. The inhibitor binds Na+,K+-ATPase reversibly and blocks ATP binding sites at micromolar concentrations with an I50 of approximately 700 nm. As a result, formation of the phosphorylated intermediate of Na+,K+-ATPase is hindered in the presence of the inhibitor. It does not affect p-nitrophenylphosphatase activity. Tryptophan fluorescence studies and CD analysis suggest conformational changes of Na+,K+-ATPase on binding to the inhibitor.     

Properties of membrane bound ferrochelatase purified from baboon liver mitochondria

1. Baboon ferrochelatase was purified to apparent homogeneity. 2. The pH optimum was 7.85 and the pI 5.3. 3. The estimated molecular weight was 205 K made up by two 50 + 60 K heterodimers. 4. The Km values for proto- and mesoporphyrin were 18.5 and 10.8 microM with iron as co-substrate. With cobalt as co-substrate the Km values were 34.5 and 10.4 microM, respectively. The mean Km value for iron was 2.2 microM while cobalt acted as a complete inhibitor. 5. Lead played a dual role that of both pseudo substrate and inhibitor. As shown by inhibitor kinetics, Pb acted as a two-step two-site parabolic competitive inhibitor. The mean Ki value at low Pb levels was 0.65 mM and at high levels 0.17 mM. 6. Substrate inhibition occurred above 36 microM for proto- and 44 microM for mesoporphyrin with iron as co-substrate. For iron, with mesoporphyrin as co-substrate it occurred above 29 microM.     

Purification and characterization of a bovine colostrum low molecular weight cysteine proteinase inhibitor

Large amounts of cysteine proteinase inhibitors were found in bovine colostrum. One had a molecular weight of 90,000, and the other a molecular weight of 10,500. The concentrations of both these inhibitors were highest the day after parturition, and were about one-tenth as much on day 7. The lower molecular weight inhibitor was purified by acid treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-50, CM-Sephadex chromatography and rechromatography on Sephadex G-50. The purified preparation gave a single band on SDS-polyacrylamide gel electrophoresis. This inhibitor contained one tryptophanyl residue and one cystinyl residue, and did not contain a free thiol group. Values obtained for its isoelectric point (pI) were 10.0 and 10.3. This material strongly inhibited cathepsin B, cathepsin H, and papain. the higher molecular weight inhibitor was partially purified. It had a pI of 4.2 and inhibited papain, cathepsin H, and cathepsin B.     

Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae).

A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1-11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of alpha-thrombin (K(i) = 4.89 +/- 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for gamma-thrombin (K(i) = 22.3 +/- 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin.     

Inactivation of TEM-1 beta-lactamase by 6-acetylmethylenepenicillanic acid

6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases. Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated. 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1. The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin. Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15. Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme. All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.     

Amino acid sequence and disulfide bridges of affinity purified Kunitz-type chymotrypsin inhibitor from winged bean seed (Psophocarpus tetragonolobus (L.) DC)

The primary sequence of the affinity purified chymotrypsin inhibitor, WBCI, isolated from the albumin fraction of Psophocarpus tetragonolobus (L.) DC cv. UPS-122 seed was determined. The inhibitor consisted of a single polypeptide chain of 183 amino acids (Mr 20285) and the four half-cystine residues in the molecule formed two intramolecular disulfide bridges equivalent to those in other Kunitz-type seed inhibitors. The sequence of this chymotrypsin inhibitor was identical to that of chymotrypsin inhibitor-3 from cultivar UPS-31 and it showed about 50% sequence similarity to the winged bean acidic (WBTI-2, pI 5.1) and basic (WBTI-1, pI 8.9) trypsin inhibitors. Sequence similarities to other Kunitz-type seed inhibitors are discussed.     

Purification of a new extracellular 90-kDa serine proteinase with isoelectric point of 3.9 from Bacillus subtilis (natto) and elucidation of its distinct mode of action.

A new extracellular 90-kDa serine proteinase with an isoelectric point (pI) of 3.9 was purified from Bicillus subtilis (natto). Microheterogeneity was detected in the 50-kDa protease of bacillopeptidase F with pI 4.4 reported previously by Wu et al. and the sequence for the first 25 amino acids in the internal region of the enzyme was analyzed: ATDGVEWNVDQIDAPKAWALGYDGA. The cleavage sites in the oxidized B-chain of insulin by the proteinase were CySO3H7-Gly8, Val12-Glu13, Try16-Leu17, and Phe25-Tyr26. The activity was inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin, while the activity was not inhibited by proteinaceous Streptomyces subtilisin inhibitor (SSI) or alpha 2-macroglubulin.     

Digestive proteinases of yellow mealworm (<Emphasis Type="Italic">Tenebrio molitor</Emphasis>) larvae: Purification and characterization of a trypsin-like proteinase

A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI 7.4. The enzyme was also characterized by temperature optimum at 55 degrees C, pH optimum at 8.5, and K(m) value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0-9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N-terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50-72% identity with other insect trypsin-like proteinases, and 44-50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.     

Characteristics of an inhibitor of the Na+/K+ pump in human cerebrospinal fluid

Human cerebrospinal fluid (CSF) inhibits the Na+/K+ pump in human red cells and the activity of purified Na+/K+-ATPase (Halperin, J. A., Shaeffer, R., Galvez, L., and Malavé, S. (1985) Proc. Natl. Acad. Sci. U.S. A. 80, 6102-6104, 1983; Halperin, J. A., Martin, A. M., and Malavé, S. (1985) Life Sci. 37, 561-566. We describe here some properties of the CSF inhibitor of the Na+/K+ pump. Active material was extracted from human CSF with 50% methanol and then concentrated and desalted by ultrafiltration. This extract inhibited, in a dose-dependent manner, the ouabain-sensitive influx of K+ into human red cells and the activity of purified Na+/K+-ATPase. Partial separation of the inhibitory activity was achieved by gel filtration and reverse-phase high performance liquid chromatography. Inhibition of both pump and enzyme was specific in that other red cell membrane transport systems or enzymes examined were not influenced by CSF extracts. Dialysis and ultrafiltration experiments indicate that the molecular weight of the inhibitor is approximately equal to 600. The inhibitory activity is sensitive to proteolytic enzymes indicating that the inhibitor might be a small peptide. In the presence of CSF extract the K0.5 for external K+ to stimulate the Na+/K+ pump increased from 1.4 to 3.1 mM, suggesting that the CSF inhibitor competes with external K+ for stimulation of the pump. We estimate that the concentration of the inhibitor in CSF might be approximately equal to 50 pg/ml, a value close to the concentration of other active peptides found in human CSF.     

Toxicological studies of the carbamates methomyl and bendiocarb in the bulb mite Rhizoglyphus echinopus (Acari: Acaridae)

Methomyl was 15 and 31.3 times more toxic than bendiocarb to bulb mites at the LC50 and LC90 values respectively. However, methomyl (pI50 3.0) was at least 126 times less active than bendiocarb (pI50 5.1) as an inhibitor of bulb mite cholinesterase in vitro. The disparity between the high toxicity of methomyl and its extremely low activity as an inhibitor of mite cholinesterase in vitro indicated that another mechanism was likely involved in its toxic action. Pharmacokinetic studies of methomyl and bendiocarb showed that penetration and metabolism were rapid and that there were no substantial differences in the internal levels of the respective parent carbamates during the 24 h test period. However, volatile radioactive material(s), some of which was carbon dioxide, was produced in appreciably greater amounts from methomyl than from bendiocarb. We speculate that the production of volatiles, such as carbon dioxide, acetonitrile and/or methylamine, may contribute to the toxicity of methomyl to bulb mites. © Rapid Science Ltd. 1998